Cross-talk between integrins alpha1beta1 and alpha2beta1 in renal epithelial cells

The collagen-binding integrins α1β1 and α2β1 have profoundly different functions, yet they are often co-expressed in epithelial cells. When both integrins are expressed in the same cell, it has been suggested that α1β1 negatively regulates integrin α2β1-dependent functions. In this study we utilized murine ureteric bud (UB) epithelial cells, which express no functionally detectable levels of endogenous integrins α1β1 and α2β1, to determine the mechanism whereby this regulation occurs. We demonstrate that UB cells expressing integrin α2β1, but not α1β1 adhere, migrate and proliferate on collagen I as well as form cellular cords in 3D collagen I gels. Substitution of the transmembrane domain of the integrin α2 subunit with that of α1 results in decreased cell adhesion, migration and cord formation. In contrast, substitution of the integrin α2 cytoplasmic tail with that of α1, decreases cell migration and cord formation, but increases proliferation. When integrin α1 and α2 subunits are co-expressed in UB cells, the α1 subunit negatively regulates integrin α2β1-dependent cord formation, adhesion and migration and this inhibition requires expression of both α1 and α2 tails. Thus, we provide evidence that the transmembrane and cytoplasmic domains of the α2 integrin subunit, as well as the α1 integrin subunit, regulate integrin α2β1 cell function.     

MARINE‐DERIVED DINOFLAGELLATES IN ANTARCTIC SALINE LAKES: COMMUNITY COMPOSITION AND ANNUAL DYNAMICS1*[link]

The saline lakes of the Vestfold Hills in Antarctica offer a remarkable natural laboratory where the adaptation of planktonic protists to a range of evolving physiochemical conditions can be investigated. This study illustrates how an ancestral marine community has undergone radical simplification leaving a small number of well‐adapted species. Our objective was to investigate the species composition and annual dynamics of dinoflagellate communities in three saline Antarctic lakes. We observed that dinoflagellates occur year‐round despite extremely low PAR during the southern winter, which suggests significant mixotrophic or heterotrophic activity. Only a small number of dominant dinoflagellate species were found in each lake, in contrast to the species‐rich Southern Ocean from which the lake communities are believed to be derived. We verified that the lake species were representatives of the marine polar dinoflagellate community, and not freshwater species. Polarella glacialis Montresor, Procaccini et Stoecker, a bipolar marine species, was for the first time described in a lake habitat and was an important phototrophic component in the higher salinity lakes. In the brackish lakes, we found a new sibling species to the brackish‐water species Scrippsiella hangoei (J. Schiller) J. Larsen, previously observed only in the Baltic Sea.     

Folinic acid attenuates methotrexate chemotherapy-induced damages on bone growth mechanisms and pools of bone marrow stromal cells

Chemotherapy often induces bone growth defects in pediatric cancer patients; yet the underlying cellular mechanisms remain unclear and currently no preventative treatments are available. Using an acute chemotherapy model in young rats with the commonly used antimetabolite methotrexate (MTX), this study investigated damaging effects of five once-daily MTX injections and potential protective effects of supplementary treatment with antidote folinic acid (FA) on cellular activities in the tibial growth plate, metaphysis, and bone marrow. MTX suppressed proliferation and induced apoptosis of chondrocytes, and reduced collagen-II expression and growth plate thickness. It reduced production of primary spongiosa bone, volume of secondary spongiosa bone, and proliferation of metaphyseal osteoblasts, preosteoblasts and bone marrow stromal cells, with the cellular activities being most severely damaged on day 9 and returning to or towards near normal levels by day 14. On the other hand, proliferation of marrow pericytes was increased early after MTX treatment and during repair. FA supplementation significantly suppressed chondrocyte apoptosis, preserved chondrocyte proliferation and expression of collagen-II, and attenuated damaging effects on production of calcified cartilage and primary bone. The supplementation also significantly reduced MTX effects on proliferation of metaphyseal osteoblastic cells and of bone marrow stromal cells, and enhanced pericyte proliferation. These observations suggest that FA supplementation effectively attenuates MTX damage on cellular activities in producing calcified cartilage and primary trabecular bone and on pools of osteoblastic cells and marrow stromal cells, and that it enhances proliferation of mesenchymal progenitor cells during bone/bone marrow recovery.     

Genetic differentiation among European whitefish ecotypes based on microsatellite data

The amount of genetic differentiation at DNA microsatellite loci in European whitefish (Coregonus lavaretus) was assessed among ecotypes, populations and run-timing types. The magnitude of genetic changes potentially caused by hatchery broodstock rearing were also compared with those observed in corresponding natural populations. A total of 35 populations were studied, including 33 Coregonus lavaretus populations and two samples of Coregonus peled. Five of the six whitefish ecotypes in Finland were represented within C. lavaretus populations. Genetic diversity among C. lavaretus populations proved to be high compared to two C. peled populations. The genetic D(A) distance between these two species was as high as 0.86. The genetic differentiation among ecotypes was generally low and thus gives support for the hypothesis of one native European whitefish species in Scandinavia. Among the ecotypes the northern, large sparsely-rakered, bottom-dwelling whitefish was most unique. Thus, observed genetic differences in quantitative traits have either developed independently of phylogenetic lineages, or have mixed and later changed according to environments and selection pressures. Overall genetic distances between the anadromous whitefish populations along the Finnish coast, especially in the Bothnian Bay area, were small. Populations of this area have been heavily influenced by human activities, and they also have the highest probability of mixing by natural means. In two cases, the Rivers Iijoki and Tornionjoki, statistically significant genetic differences could be observed between summer- and autumn-run spawning-time types. Wild populations had slightly higher allelic diversity than hatchery-reared populations of corresponding rivers. Although some reduction in genetic diversity during hatchery rearing is possible, it is an important aid in maintaining endangered populations.     

Deciphering the ligand-binding sites in the Borrelia burgdorferi complement regulator-acquiring surface protein 2 required for interactions with the human immune regulators factor H and factor H-like protein 1

Borrelia burgdorferi, the etiologic agent of Lyme disease, employs sophisticated means to evade killing by its mammalian hosts. One important immune escape mechanism is the inhibition of complement activation mediated by interactions of the host-derived immune regulators factor H (CFH) and factor H-like protein 1 (CFHL1) with borrelial complement regulator-acquiring surface proteins (BbCRASPs). BbCRASP-2 is a distinctive CFH- and CFHL1-binding protein that is produced by serum-resistant B. burgdorferi strains. Here we show that binding of CFH by BbCRASP-2 is due to electrostatic as well as hydrophobic forces. In addition, 14 individual amino acid residues of BbCRASP-2 were identified as being involved in CFH and CFHL1 binding. Alanine substitutions of most of those residues significantly inhibited binding of CFH and/or CFHL1 by recombinant BbCRASP-2 proteins. To conclusively define the effects of BbCRASP-2 residue substitutions on serum sensitivity in the bacterial context, a serum-sensitive Borrelia garinii strain was transformed with plasmids that directed production of either wild-type or mutated BbCRASP-2 proteins. Critical amino acid residues within BbCRASP-2 were identified, with bacteria producing distinct mutant proteins being unable to bind either CFH or CFHL1, showing high levels of complement components C3, C6, and C5b-9 deposited on their surfaces and being highly sensitive to killing by normal serum. Collectively, we mapped a structurally sensitive CFH/CFHL1 binding site within borrelial BbCRASP-2 and identified single amino acid residues potentially involved in the interaction with both complement regulators.     

Phosphorylation of murine caspase-9 by the protein kinase casein kinase 2 regulates its cleavage by caspase-8

Previous studies from our laboratory had indicated that cytochrome c-independent processing and activation of caspase-9 by caspase-8 contributed to early amplification of the caspase cascade in tumor necrosis factor (TNF)-alpha-treated murine cells. Here we show that murine caspase-9 is phosphorylated by casein kinase 2 (CK2) on a serine near the site of caspase-8 cleavage. CK2 has been shown to regulate cleavage of the pro-apoptotic Bid protein by phosphorylating serine residues near its caspase-8 cleavage site. Similarly, CK2 modification of Ser(348) on caspase-9 appears to render the protease refractory to cleavage by active caspase-8. This phosphorylation did not affect the ability of caspase-9 to autoprocess. Substitution of Ser(348) abolished phosphorylation but not cleavage, and a phospho-site mutant promoted apoptosis in TNF-alpha-treated caspase-9 knock-out mouse embryo fibroblasts. Furthermore, inhibition of CK2 activity and RNA interference-mediated knockdown of the kinase accelerated caspase-9 activation, whereas phosphatase inhibition delayed both caspase-9 activation and death in response to TNF receptor occupation. Taken together, these studies show that TNF receptor cross-linking promotes dephosphorylation of caspase-9, rendering it susceptible to processing by activated caspase-8 protein. Thus, our data suggest that modification of procaspase-9 to protect it from inappropriate cleavage and activation is yet another mechanism by which the oncogenic kinase CK2 promotes survival.     

A Mouse Surgical Model for Metastatic Ovarian Granulosa Cell Tumor

We recently described a genetically engineered mouse model that develops ovarian granulosa cell tumors (GCTs) that mimic many aspects of the advanced human disease, including distant dissemination. However, because the primary tumors killed their hosts before metastases were able to form, the use of these mice to study metastatic disease required the development of a simple, reliable, and humane surgical protocol for the excision of large GCTs from debilitated mice. Here we describe a protocol involving multimodal anesthesia, tumor removal through ventral midline celiotomy and perioperative fluid therapy, and analgesia that led to the postoperative survival of more than 90% of mice, despite the removal of tumors representing as much as 10% of the animal''s body weight. Intraabdominal recurrence of the GCT did not occur in surviving animals, but most developed pulmonary or adrenal metastases (or both) by 12 wk after surgery. We propose that this mouse model of metastatic GCT will serve as a useful preclinical model for the development of novel treatment modalities and diagnostic techniques. Furthermore, our results delineate anesthetic and surgical principles for the removal of large abdominal tumors from mice that will be applicable to other models of human cancers.Abbreviation: GCT, granulosa cell tumorThe ovarian granulosa cell tumor (GCT) is the most prevalent sex cord tumor in women and is thought to represent up to 5% of all ovarian cancers.⁸ It is generally considered a low-grade malignancy, but a small percentage of GCTs are considered aggressive, and a large proportion of patients develop recurrent disease postoperatively.¹⁰ Few therapeutic options are available for the recurrent disease, and fastidious long-term follow-up is required in most patients, given that recurrent lesions have been diagnosed as long as 40 y after removal of the original tumor.⁴One of the key factors that has limited progress in the development of therapeutic approaches for ovarian cancer is the dearth of relevant animal models.⁹ Our laboratory has recently developed a genetically engineered mouse model (Pten^{tm1Hwu/tm1Hwu}; Ctnnb1^tm1Mmt/+;Amhr2^{tm3(cre)Bhr/+}) that develops an aggressive form of GCT in animals of both sexes.¹ In female mice, the disease is characterized by perinatal onset and rapid tumor development in both ovaries, causing death by approximately 8 wk of age. Histopathologic analyses of 6- to 8-wk-old Pten^{tm1Hwu/tm1Hwu}; Ctnnb1^tm1Mmt/+; Amhr2^{tm3(cre)Bhr/+} tissues revealed the presence of tumor cell embolisms in the lungs, suggesting metastatic potential for GCTs.⁶ However, the mice invariably died or required euthanasia for humane reasons before true metastases were able to form. Surgical excision of the primary tumors allowed for the postoperative survival of the mice for several months, and permitted the development of metastases in several tissues including lung and adrenal gland, thereby confirming the metastatic potential of the tumor cells.⁶Because of its mimicry of advanced human disease from the molecular to tumor biology level, we have proposed that the Pten^{tm1Hwu/tm1Hwu};Ctnnb1^tm1Mmt/+;Amhr2^{tm3(cre)Bhr/+} model could represent a powerful tool for the development of new therapeutic strategies for metastatic GCT. However, because the GCTs kill their hosts before metastases are able to form, their use as a model of the metastatic disease requires the development of a simple, reliable, and humane surgical protocol for the excision of the primary tumors. Such a protocol would need to take into account that pulmonary tumor cell embolisms have not been noted in these mice before the age of 6 wk, necessitating that surgery be performed in debilitated animals with tumor burdens approaching 10% of their body weight. A review of the existing literature on anesthesia, fluid therapy, pain management, and surgical techniques in mice yielded little information applicable to our model, necessitating that we develop a novel protocol that addressed both the medical needs of the mice and the scientific objectives of the research program.